This invention relates to the fields of antibodies and fragments thereof, immunology, biological and chemical assay development, drug discovery, medical diagnostics and treatments, and proteomics.
Andrew, S. M, and Titus, J. A. (1997). Purification and Fragmentation of Antibodies. In Current Protocols in Immunology, edited by Coligan, J. W., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. and Strober, W., John Wiley and Sons, N.Y., pp. 2.7.1-2.7.12.
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Parham, P. (1986). Preparation and purification of active fragments from mouse monclonal antibodies. In Handbook of Experimental Immunology, Vol. 1: Immunochemistry (D. M. Wier, ed.) pp14.1-14.23. Blackwell Scientific, Oxford.
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Antibodies, and in particular, antibody fragments, are heavily utilized in diagnostic, therapeutic, and biological research applications. Often there are substantial advantages to using antibody fragments that are produced by proteolysis of IgGs.
Full size IgG antibodies have three domains, each of approximately 50 kd molecular weight, the three domains comprising two identical xe2x80x9cFabxe2x80x9d (antigen binding) fragments, and an Fc (crystallizable domain). It is often advantageous to remove the Fc domain from the antibody prior to use to yield, as in the case of pepsin cleavage, a F(abxe2x80x2)2 fragment separated from the Fc domain. An F(abxe2x80x2)2 maintains the binding characteristics of a full size IgGs despite its loss of the Fc domain. The Fc domain can invoke a variety of undesired biological effector functions that can interfere with the therapeutic or diagnostic uses of the antibodies, thus removal of the Fc region has substantial value. The F(abxe2x80x2)2 is also a useful intermediate in the production of monomeric, chemically tagged Fab monomers because F(abxe2x80x2)2s are held together by 1-3 disulfide bonds between the heavy chains. Mild chemical reduction of such disulfide bonds may result in the formation of monomeric Fab fragments having cysteines available for reacting with chemical labels or reactive surfaces.
Several classes of IgG antibodies exist having differences based on the sequence of the heavy chain. Consequently, different classes have different susceptibilities to proteolysis by pepsin. Mouse-derived monoclonal antibodies include four IgG subclasses: 1, 2a, 2b and 3. Certain, and often important, members of antibody classes 1 and 2b are recalcitrant to yielding F(abxe2x80x2)2 fragments from pepsinolysis treatment. Even if pepsin cleaves such antibodies, it often does not give good yields or yields different non-F(abxe2x80x2) 2 products. Thus many important IgGs cannot be efficiently converted to Fab dimers. Because IgG1 class is the most common for monoclonal antibodies used in biotechnology, there is a need for reliable, universal methods for converting whole IgG1 and other pepsin resistant antibodies to intact F(abxe2x80x2)2 antibody fragments.
Methods for the preparation of F(abxe2x80x2)2 fragments by pepsinolysis have been described which produce antibody fragments that retain full binding activity but do not possess the effector functions conferred by the Fc domain. See Nisonoff et al., and Andrew and Titus. However, as discussed herein, these methods are of limited use depending, in part, on the type and source of antibody used as a starting material. F(abxe2x80x2)2 fragments may be selectively reduced to Fab fragments having free cysteines in the linker region (Nisonoff et al.) This allows Fab fragments to be labeled or attached to solid supports or labels through a region of the protein that is distal to the antigen-binding site. The most common method for generating F(abxe2x80x2)2 fragments is by pepsinolysis, which is generally efficient for most antibodies from the mouse IgG2a and IgG3 subclasses, but not generally efficient for those from the IgG2b or IgG1, the latter being the most common.
Many others have reported poor yields of F(abxe2x80x2)2 fragments by treating mouse IgG1 antibodies with pepsin under standard conditions (37xc2x0 C., pH 4.5), and such procedures typically also produce several other cleavage products as well (See Gorini et al.; Laymoyi and Nisonoff; Parham; Mariani et al.; and, Andrew and Titus.) About 50% of the IgG1 antibodies appeared to be completely resistant to pepsinolysis. Numerous alternatives to pepsinolysis have been described for generating F(abxe2x80x2)2 fragments from IgG1 molecules, including the use of papain (under slightly reducing conditions), V8 protease, or ficin, for example. See generally Parham; Milenic et al.; Mariana et al.; Yamaguchi et al.; and, Andrew and Titus, however, each of these failed to provide a reliable method for preparing F(abxe2x80x2)2s from antibodies with uniform, predictable results. Thus, there is a need for a universal method for preparing F(abxe2x80x2)2 antibody fragments from whole antibodies, especially those from IgG1 and IgG2b subclasses. There is also a need for a method for converting other immunoglobulins from other species such as chickens and their IgY antibodies. The invention disclosed herein addresses these, and other needs as discussed below and as will become apparent to one of ordinary skill in the art reading this disclosure and subsequent claims.
The invention provides methods for making F(abxe2x80x2)2 antibody fragments from antibodies, in particular, antibodies that have one or more oligosaccharide groups attached to regions of the antibody other than the hinge region.
In one aspect, the invention provides a method for preparing a F(abxe2x80x2)2 fragment from a glycosylated antibody. The method includes the steps of providing a glycosylated antibody where the glycosylated antibody has a hinge region having one or more protease cleavage sites located within the hinge region, and one or more non-hinge regions adjacent the hinge region, the non-hinge region(s) having one or more oligosaccharide groups attached thereto, where the oligosaccharide group(s) cause the protease cleavage site(s) within the hinge region to be resistant to a proteolysis treatment. The glycosylated antibody or antibodies are then exposed to a deglycosylation treatment, the deglycosylation treatment cleaving the oligosaccharide group(s) attached to the non-hinge region(s) to form a partially or wholly deglycosylated antibody having a hinge region cleavable by the proteolysis treatment. The partially or wholly deglycosylated antibody or antibodies are then exposed to the proteolysis treatment to cause proteolytic cleavage of the hinge region cleavable by the proteolysis to form the F(abxe2x80x2)2 fragment.
Certain preferred embodiments may have at least one of the following features such as; the glycosylated antibody being a plurality of glycosylated antibodies, at least some of the glycosylated antibodies being polyclonal, the glycosylated antibodies being monoclonal, the glycosylated antibody being either an IgG1 or IgG2b glycosylated antibody, the IgG1 or IgG2b antibody being from a rodent-derived hybridoma cell culture or ascites, the glycosylated antibody being derived from the group consisting of rat, mouse, rabbit, goat, sheep, lamb, chicken, or horse, the proteolysis being achieved wholly or partly from protease treatments including components selected from the list consisting of pepsin, proteases that cleave pepsin substrates, papain, papain pre-activated with cysteine, and ficin, the proteolysis being achieved by a protease capable of producing F(abxe2x80x2)2 fragments from the deglycosylated antibodies, the deglycosylase treatment containing a glycosidase combination selected from the group consisting of PNGase F, endo-O-glycosylase, sialidase A, PNGase F/endo-O-glycosylase, PNGase F/sialidase A, PNGase F/endo-O-glycosylase/sialidase A, endo-O-glycosylase/sialidase A, and/or the non-hinge regions comprising the Fc and the F(abxe2x80x2) regions of the glycosylated antibody.
Another aspect of the invention provides for a method for preparing F(abxe2x80x2)2 fragments. The method includes the steps of growing a hybridoma cell that normally produces glycosylated antibodies where the glycosylated antibodies have a hinge region with one or more protease cleavage sites located within the hinge region, one or more non-hinge regions adjacent the hinge region, and one or more oligosaccharide groups being attached to at least one of the non-hinge regions by the hybridoma cell through glycosylation, the oligosaccharide groups causing the hinge regions to be resistant to a proteolysis treatment. The hybridoma cell or cells are administered an inhibitor of the glycosylation effective to inhibit glycosylation of the antibodies to produce one or more unglycosylated antibodies lacking the oligosaccharides within at least one non-hinge region to render the hinge region prone to the proteolysis treatment. The unglycosylated antibodies are exposed to the proteolysis treatment so that the unglycosylated antibodies"" binge regions are cleaved to form the F(abxe2x80x2)2 fragments from the unglycosylated antibodies.
Certain preferred embodiments of the invention may include the hybridoma cell being a part of a hybridoma cell culture or ascites, the hybridoma cell being a plurality of hybridoma cells, the hybridoma cell being part of a monoclonal or polyclonal hybridoma cell line, the hybridoma cells being from the same hybridoma cell line, the hybridoma cells being from different hybridoma cell lines, and/or the inhibitor of the glycosylation contains bacitracin or tunicamycin.
Another aspect of the invention provides a method for preparing F(abxe2x80x2)2 fragments. The method includes the steps of providing a hybridoma cell line that normally produces glycosylated antibodies, the glycosylated antibodies having a hinge region with one or more protease cleavage sites located within the hinge region, one or more non-hinge regions adjacent the hinge region, and one or more oligosaccharide groups being attached to at least one of the non-hinge regions by the hybridoma cell through glycosylation, where the oligosaccharide groups cause the hinge regions to be resistant to a proteolysis treatment. The hybridoma cell line or lines are then altered to inhibit glycosylation of the antibodies within the non-hinge regions to produce one or more unglycosylated antibodies such that the unglycosylated antibodies are susceptible to proteolysis treatment and caused to produce the unglycosylated antibodies. The unglycosylated antibodies are then exposed to the proteolysis treatment to cleave the unglycosylated antibodies to produce the F(abxe2x80x2)2 fragments.
Certain preferred embodiments may have the hybridoma cell being part of a hybridoma cell culture or ascites, and/or the altered cells being either permanently or transiently altered.
In another aspect, the invention provides for an F(abxe2x80x2)2 composition comprising: one or more F(abxe2x80x2)2 fragments, or derivative therefrom, produced by a method selected from the methods disclosed above. In certain embodiments, the F(abxe2x80x2)2 fragments are an active ingredient of an anti-toxin or anti-venom medicament.
Yet another aspect of the invention provides for an immunoglobulin composition comprising: one or more aglycosylated or deglycosylated immunoglobulins, the aglycosylated or deglycosylated immunoglobulins being formed by preventing the attachment of one or more oligosaccharides to the immunoglobulin, or effecting the removal of an attached oligosaccharide from the immunoglobulin by exposure to one or more deglycosylases, or both by preventing attachment to and removing one or more oligosaccharides from the immunoglobulin. At least one of the one or more aglycosylated or deglycosylated immunoglobulins becomes cleavable by a protease which cleaves the aglycosylated or deglycosylated immunoglobulins at a position to form F(abxe2x80x2)2 fragment(s) from the aglycosylated or deglycosylated immunoglobulins as a result of the immunoglobulin being aglycosylated or deglycosylated.
Still yet another aspect of the invention provides for a kit for making F(abxe2x80x2)2 fragments from one or more immunoglobulins, at least one of the immunoglobulins having one or more oligosaccharides attached thereto that inhibit protease activity that converts the immunoglobulins into F(abxe2x80x2)2 fragments comprising: a deglycosylation composition containing one or more deglycosylase enzymes capable of removing some or all of the oligosaccharides; and, a protease composition containing one or more proteases capable of reacting with the immunoglobulin produces F(abxe2x80x2)2 fragments from the deglycosylated antibodies. In certain preferred embodiments, the kit further comprises a purification medium for purifying the F(abxe2x80x2)2 fragments from non-F(abxe2x80x2)2 fragments of the immunoglobulin or from uncleaved immunoglobulin, and/or the kit further comprises instructions for carrying out the method selected from the group consisting of the methods disclosed above.
These and other aspects and embodiments thereof of the invention will become apparent to one skilled in the art by way of reading the specification and drawings below.